Neointima formation is a common response of arteries to injury and results, in part, from vascular smooth muscle cell (vsmc) proliferation, migration and connective tissue formation. The mechanisms by which VSMC proliferate in response to mitogenic signals are well-described; however, the role of cellular gene products which cause vsmcs to shift from a proliferative to a quiescent state during G1 phase of the cell cycle are not well-understood. The goal of this grant is to study control of VSMC cycle by p21 and p27 cyclin-dependent kinase inhibitors (CKIs). Transit through G1 and entry into the S phase requires the action of cyclin-dependent kinases (CDKs), and CDKs are inactivated by protein phosphorylation and association with regulatory subunits, including the cyclins and the CKIs. CKIs directly implicated in mitogen dependent CDK regulation are p21 and p27. In preliminary studies, they have demonstrated that p21 inhibits VSMC growth by arrest at the G1/S phase of the cell cycle and that p21 and p27 are detected in the neointima of injured arteries in vivo in a time pattern that inversely correlates with intimal cell proliferation. These findings suggest that these CKIs may normally limit the degree of intimal hyperplasia in vivo and that modulation of their expression may play a useful role in treatments for vascular diseases. On the basis of these studies, they have hypothesized that these proteins alter VSMC proliferation through their ability to regulate progression through G1 and entry into S phase of the cell cycle. To explore this hypothesis, they propose to: 1) examine the mechanism of G1 growth arrest of vsmcs by p21 and p27 in vitro; 2) determine the function of p21 and p27 in the development of atherosclerosis in p21-/-mice crossbred with apoE-/-mice and following vascular injury in p21-/-mice, p27-/-mice, and double knock-out mice; and 3) examine how p21 and p27 expression in mouse atherosclerotic lesions compares to expression of these proteins in human atherosclerotic lesions and in porcine models of balloon injury. The proposed studies should define the mechanisms by which p21 and p27 alter VSMC proliferation following vascular injury and during development of atherosclerosis through their ability to regulate cell cycle progression in G1. An understanding of these mechanisms should lend insight into the pathophysiology of vascular diseases, and determine whether enhancement of p21 and p27 expression in arteries may limit excessive vsmc proliferation in these diseases.